Acta Neurobiol Exp 2004, 64: 481-489
NEU OBIOLOGI E
EXPE IMENT LIS
Expansion of the Golgi apparatus
in rat cerebral cortex following
intracerebroventricular injections
of streptozotocin
Pawe³ Grieb1, Tomasz Kryczka1, Micha³ Fiedorowicz1,
Ma³gorzata Frontczak-Baniewicz2 and Micha³ Walski2
1
Department of Experimental Pharmacology; and 2Laboratory of Cell
Ultrastructure, Mossakowski Medical Research Centre, Polish Academy
of Sciences, 5 Pawinskiego St., 02-106 Warsaw, Poland
Abstract. Streptozotocin (STZ) is a bacterial toxin which selectively damages
both insulin-producing cells and insulin receptors. Injections of STZ into the
cerebral ventricles of experimental animals are followed by sustained
biochemical, metabolic and behavioral effects resembling those which are
found in human brains afflicted by Alzheimer’s disease. The aim of the
present study was to assess the effects of double intracerebroventricular
application of STZ on the ultrastructure of rat frontoparietal cortical neurons.
The most prominent change, seen 3 weeks after STZ injection, was
a significant enlargement of the Golgi apparatus caused by expansion of the
trans-Golgi segment of the cellular protein secretory pathway. Morphometric
analysis revealed that the area of the trans part of the Golgi complex in
3
3
neuronal cells was increased more than two-fold (median values: 312 × 10 nm
3
3
in 14 neurons from control animals, and 846 × 10 nm in 19 neurons from
STZ-treated animals, P=0.0012), whereas that of the cis part did not
significantly change. The effects of STZ did not resemble Golgi atrophy and
fragmentation described in neurons from disease-prone brain structures of
patients with Alzheimer’s disease, but were similar to that observed after
intravenous application of a non-metabolizable glucose analog
2-deoxyglucose. Considering that proamyloidogenic processing of
beta-amyloid precursor protein may occur preferentially in the trans-Golgi
segment, the observed early response of neuronal ultrastructure to
desensitization of insulin receptors may predispose cells to form beta-amyloid
deposits.
The correspondence should be
addressed to P. Grieb,
Email: [email protected]
Key words: Alzheimer’s disease, Golgi apparatus, streptozotocin
482
P. Grieb et al.
INTRODUCTION
Streptozotocin (2-deoxy-2-(3-methy1-3-nitrosoureido)-D-glucopyranose, STZ) is an antibiotic derived from
the soil bacteria Streptomyces achromogenes (Lewis
and Barbiers 1960). It had been developed as an
anticancer agent (White 1963). However, since the discovery of its diabetogenic effects following systemic application (Rakieten et al. 1963) STZ is now used mainly
to induce diabetes in experimental animals. When injected intravenously or subcutaneously, it induces diabetes resembling human type 1 (IDDM) or type II
(NIDDM) disease, depending on protocol particulars
(see for example Ito et al. 1999 and the references cited
therein). This is because STZ is toxic both to pancreatic
insulin-producing b-cells (Wilson and Leiter 1990) and
to insulin receptors (Kadowaki et al. 1984, Meyerovitch
et al. 1989), although in each case the mechanism of toxicity appears to be different. Toxicity toward b-cells most
likely involves DNA alkylation and poly(ADP)rybosylation and can be selectively blocked by nicotinamide
to create the "pure" animal model of NIDDM (Masiello et
al. 1998), while toxicity toward insulin receptors is probably mediated by free radical mechanisms.
The brain responds poorly to systemic STZ application. For example, in STZ-diabetic rats the content of insulin receptors was increased in liver, kidney and
skeletal muscle, but unchanged in neocortex (Pezzino et
al. 1996). In a recent magnetic resonance spectroscopic
study no changes in cerebral energy metabolism were
observed after up to 8 months of STZ-induced diabetes,
although some reductions in N-acetylaspartate levels in
the brain were seen indicating metabolic or functional
abnormalities in cerebral neurons (Biessels et al. 2001).
However, STZ given intracerebroventricularly (i.c.v.)
exerts profound and long-lasting influence on brain biochemistry, metabolism and function, including, inter
alia, decreased glucose uptake and energy consumption, increased lactate output, decreased phospholipid
content, tissue oxidative stress, cholinergic deafferentation, and compromised motor and cognitive performance (Duelli et al. 1994, Hoyer and Lannert 1999,
Muller et al. 1998, Prickaerts et al. 1999, Sharma and
Gupta 2001a). Collectively all these effects, which are
long-lasting, resemble those seen in humans suffering
from Alzheimer’s disease (AD), and i.c.v. STZ has been
expressis verbis proposed as a simple animal model of
sporadic AD (Hoyer et al. 2000). The results of animal
experiments with i.c.v. STZ have also been quoted as
the evidence supporting the "glucose resistance" hypothesis of AD (Hoyer 2002a,b), according to which the
dysfunction of brain insulin receptors is a primary
pathogenetic event in AD (Hoyer 2002a,b, Meier-Ruge
and Bertoni-Freddari 1996).
The "glucose resistance" hypothesis is in opposition to
the most popular "amyloid cascade" hypothesis according to which a primary pathogenetic event in AD is formation of insoluble, neurotoxic amyloid beta-peptide
(Abeta) deposits which results from imbalance between
Abeta production and catabolism (Hardy and Higgins
1992, Hardy and Selkoe 2002). Abeta is derived from
beta-amyloid precursor protein (b-APP) post-translationally modified and proteolytically cleaved within the
endoplasmic reticulum and Golgi apparatus (Mattson
1997, Selkoe 2001). In Alzheimer’s brains the Golgi apparatus of neurons from areas affected by the disease has
been found fragmented and atrophic (Stieber et al. 1996).
This, and the observation concerning linkage of a
Golgi-related gene to a very aggressive form of AD
(Sherrington et al. 1995) led to the hypothesis that disintegrated and atrophic Golgi complex may display a defect in
b-APP processing which could result in elevated production
of the amyloidogenic Abeta peptide (Dal Canto 1996).
If brain resistance to insulin is the primary
pathogenetic factor in AD, it may be a cause of fragmentation and atrophy of the Golgi complex, and Golgi
complex pathology could link impaired brain glucose
metabolism to the stimulation of amyloidogenic processing of b-APP. If this hypothesis is correct, brain insulin receptor desensitization induced by i.c.v. STZ
should also induce neuronal Golgi atrophy and fragmentation. We were unable to find any data concerning
the effects of STZ on brain cells’ Golgi morphology. Interestingly, however, STZ given systemically has been
reported to have little effect on the liver cells Golgi appearance, except of that it significantly decreased the
size of this organelle and seemed to suppress its secretory activity (Sarnecka-Keller et al. 1980, Kordowiak
1986). In the experiments reported herein we describe
the effects of i.c.v. STZ on the ultrastructural morphology of the fronto-parietal neurons, with particular emphasis on the changes of the Golgi apparatus.
METHODS
The protocol of the study was approved by the First
Ethical Committee of the City of Warsaw. The present
study was performed on outbred male Wistar rats,
i.c.v. streptozotocin and Golgi morphology 483
230-350 g body weight, bred in our facility and kept in
an air-conditioned room in 12/12 h light/dark cycle with
free access to water and food ad libitum. For
intracerebroventricular injections and brain tissue sampling the animals were anesthetized with the mixture of
ketamine (70 mg/kg) and xylazine (20 mg/ml).
Streptozotocin (STZ) purchased from Sigma was dissolved directly before use in artificial cerebrospinal
fluid to a concentration of 25 mg/ml. Rats were placed
in a stereotaxic frame and after incising skin and muscles holes were drilled bilaterally in the skull over the
lateral ventricles. STZ (0.5 mg per each side) or vehicle
was injected to the vicinity of each lateral ventricle
(0.8 mm caudally from bregma, 1.5 mm laterally from
sutura sagittalis, 3.6 mm below the brain surface) with
the use of a Hamilton micro-syringe. The volume injected was 20 µl/ventricle. Holes in the skull were secured with a bone wax and the skin was sutured. The
stereotaxically-guided injections were repeated after
3 days. To avoid infections the rats were prophylactically treated with ceftriaxon (0.2 g per day).
Three weeks after the first injection of STZ or vehicle
the animals were perfused via the left heart ventricle
with a fixative consisting of 2% paraformaldehyde and
2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4
at 20°C. Tissue for ultrastructural studies was sampled
from the fronto-parietal lobe of cerebral cortex, approx.
5 mm rostrally from the injection site (Fig. 1). This area
was chosen because, according to Duelli and coauthors
Fig. 1. A scheme showing the distance between the injection
site and the site of sampling brain cortex for electron microscopy
(1994), in rat brain following i.c.v. STZ injection, glucose consumption is most severely affected in frontal
and in parietal cortex. The tissues were fixed for 20 h,
postfixed in a mixture of 1% OsO4 and 0.8%
K4Fe(CN)6, and processed for transmission electron microscopy. After dehydration in series of ethanol and
propylene oxide, tissue specimens were embedded in
Spurr resin. Ultrathin (50 nm) sections were examined
with a JEM 1200EX electron microscope.
To quantify the effect of treatment on the Golgi
complex, morphometric analysis was performed
blindly on brain cortical tissue samples from 3 control
and 3 STZ-treated rats; for comparison, brain cortical
Fig. 2. Delineation of the cis and the trans Golgi. Upper panel:
a micrograph of the Golgi in a neuronal cell at the high magnification. Lower panel: a sketch of the contours of organellae
seen in the micrograph above. Broken line with arrows is the
outline of the cis and the trans part of the Golgi, which consist
of the long and parallel cisterns and of less regular and shorter
tubules and vesicles described in literature as the saccotubular
network, respectively. (L) lysosome; (ER) endoplasmic reticulum; (R) ribosomes.
484
P. Grieb et al.
tissue from 3 rats treated with 2-deoxyglucose as described in the preceding paper (Grieb et al. 2004) have
also been analyzed. Tissue blocks with regions of
well-preserved Golgi were returned to the microtome
and ribbons of 5-6 serial 50 nm sections were cut. Each of
these sections were then micrographed at high magnification (50 000 ×). A Microscan software package for the
analysis of microscopic pictures (Centrum Mikroskopii, Warsaw, Poland) was used for determination of
the size of the Golgi system in the neurons. On each micrograph the contours of the cis (i.e., proximal) and
trans (i.e., distal) part of the Golgi complex were outlined as shown on Fig. 2, and their respective areas (expressed in square nanometers) were determined. The
data for each series of sections were averaged and analyzed further as a single result concerning a given neuron. The Golgi complexes in 14 neurons from control
animals, in 19 neurons from STZ-treated and in 25 neurons from 2-DG-treated animals were measured. Statistical analysis of the morphometric data was performed
using Statistica 6 (Statsoft) software package, taking
P<0.05 as the indication of significance.
RESULTS
In the fronto-parietal cortex samples taken from control rats a normally appearing brain tissue was found including neurons with visible Golgi apparatuses in which
Fig. 3. Three weeks after the first i.c.v. injection of STZ. The
perikaryal parts of the neuron. A single long endoplasmic reticulum (arrow heads) is seen in the vicinity of the nucleus. In
cytoplasm aggregates of ribosomes, and inclusions resembling deposits of lipofuscin are present (arrows). Magnification, × 12 000.
Fig. 4. Three weeks after the first i.c.v. injection of STZ. The
part of the neuronal cell with an expanded Golgi system. An
irregular dilatation of cisternae and aggregation of smooth
microvesicles in the vicinity of the Golgi complex are seen.
Magnification, × 50 000.
the cis and trans parts were most frequently of similar
size. In the fronto-parietal cortex samples taken from
rats injected intracerebroventricularly with streptozotocin characteristic morphological alterations in cellular organelle of the neurons prevailed. Endoplasmic
reticulum was characterized by long, single cisternae
present in the perinuclear zona (Fig. 3). The cisternae
were devoid of ribosomes, which were dispersed in
other parts of the cell. The mitochondria were not
ultrastructurally changed. Cytoplasmatic inclusions resembling deposits of lipofuscin were frequently encountered (Fig. 4).
The most prominent ultrastructural feature of the cortical neurons from the cortex of i.c.v. STZ-treated rats
Fig. 5. Three weeks after the first i.c.v. injection of STZ. In the
vicinity of nucleus the Golgi complex with characteristically
expanded trans-Golgi network is present. Magnification,
× 24 000.
i.c.v. streptozotocin and Golgi morphology 485
was ultrastructural alterations of the Golgi apparatus.
They were characterized by an increase in the number
and size of cisternae and their irregular dilatation. In
the cis face of the Golgi complex the single cisternae
were dilated. Aggregates of smooth microvesicles
were present in the cis and trans face. The trans part
of the Golgi complex was visibly expanded, presenting itself as a large saccotubular network (Fig. 5). No
signs of Golgi atrophy in cortical neurons were ever
seen.
Results of the morphometric analysis are presented in
Table I. Because in all but 4 cases the distribution of variables was significantly different from normal (Shapiro-Wilks P<0.05), the non-parametric Kruskal-Wallis
ANOVA test was employed to evaluate significance of
differences. With the exception of the area of the cis part
of the Golgi complex which did not change due to treatments, all treatment effects were significant, indicating
a prominent expansion of the trans (i.e., distal) part of
this organellum. However, the effects of streptozotocin
were much more pronounced than the effects of
2-deoxyglucose.
DISCUSSION
In our experiments the site of STZ injection was relatively distant from the site of cortical tissue sampling for
EM (see Fig. 1). One may argue that the toxin injected
into the cerebral lateral ventricles may act locally rather
than diffuse across the whole bulk of the brain. Although such a possibility cannot be excluded, decomposition of STZ in biological milieu may involve generation
of nitric oxide (Kwon et al. 1994), methyldiazohydroxide
and alkylating methyldiazonium and carbonium cations
(Wilson and Leiter 1990). It seems conceivable that
these highly diffusible metabolites easily penetrate
across brain extracellular spaces and reach locations
distant from the injection site.
Metabolic and behavioral disturbances which follow
single or double i.c.v. STZ injection(s) are long-lasting.
Table I
The size of the Golgi apparatus and its subcompartments in cerebral cortical neurons
Treatment
(number of neurons evaluated)
Control
(n=14)
2-DG
(n=25)
STZ
(n=19)
832 (609)
580 (401; 1 287)
No (P<0.02)*
961 (378)
907 (691; 1 211)
Yes
Treatment effect: P=0.0174**
1 402 (813)
1 249 (777; 1 556)
No (P<0.005)
366 (262)
258 (206; 455)
No (P<0.02)
317 (122)
284 (205; 405)
Yes
Treatment effect: ns
439 (303)
369 (227; 538)
No (P<0.006)
Trans part of Golgi (× 10 nm )
Mean (SD)
Median (lower quart.; upper quart.)
Normal distribution
467 (382)
312 (193; 716)
No (P<0.02)
645 (304)
524 (481; 799)
No (P<0.003)
Treatment effect: P=0.0012
962 (529)
846 (565; 1 018)
No (P<0.006)
Trans/Cis ratio of areas
Mean (SD)
Median (lower quart.; upper quart.)
Normal distribution
1.27 (0.44)
1.25 (0.93; 1.53)
Yes
2.17 (0.92)
1.96 (1.58; 2.55)
No (P<0.003)
Treatment effect: P=0.0001
2.50 (0.89)
2.13 (1.89; 3.11)
Yes
3
2
Whole Golgi (× 10 nm )
Mean (SD)
Median (lower quart. upper quart.)
Normal distribution
3
2
Cis part of Golgi (× 10 nm )
Mean (SD)
Median (lower quart.; upper quart.)
Normal distribution
3
2
*Shapiro-Wilk’s test; **Kruskal-Wallis ANOVA
486
P. Grieb et al.
This can be explained by the inability of brain tissues to
fully compensate for the damage inflicted to the insulin
receptors by the toxin or its metabolite(s). However, the
presence of tissue oxidative stress in brain 3 weeks after
i.c.v. STZ (Sharma and Gupta 2001a) and the ability of
various antioxidants to ameliorate the effects of i.c.v.
STZ (Sharma and Gupta 2001b, 2002, Veerendra
Kumar and Gupta 2003) cannot be directly related to the
injection of the toxin which is supposed to act transiently and extracellularly. In diabetes mellitus tissue
oxidative stress has been attributed to hyperglycemia,
but this explanation does not apply to the case of STZ injected into the cerebral ventricles, because systemic
glycemia is not affected. Sustained oxidative stress in
brain tissues (Sharma and Gupta, 2001a) and cytoplasmatic lipofuscin-like deposits which we have found
following i.c.v. injections of STZ (free radical-mediated
mechanisms are implicated in the generation of
lipofuscin (Brunk and Terman 2002)) seem to reflect
changes in brain metabolism which somehow result in
excessive generation of free radicals.
When neurons degenerate, it seems rather obvious
that their metabolic activity, including activity of the
Golgi apparatus, would be diminished. Therefore, the
atrophy and fragmentation of this organelle seen in neurons from human AD-afflicted brains (Stieber et al.
1996) may reflect the end-stage of the disease. In a series of papers (Salehi et al. 1994, 1995a,b,c, 1998) summarized in a review article of Salehi and Swaab (1999)
extensive data concerning neurons of young and old AD
patients as well as young and old controls were presented. These authors found no relationship between the
size of the Golgi complex and age or severity of the
cytoskeletal alterations in the neurons from nucleus
tuberalis. However, in nucleus basalis of Meynert as
well as in tuberomammilary neurons and in the CA1
sector of hippocampus the size of Golgi apparatus in AD
patients was smaller than in controls. These data suggest
that in AD there is reduced protein processing in neurons located in disease-affected areas, although it does
not directly correlate with neuropathologic hallmarks of
the disease such as neurofibrillary tangles. However, in
vasopressin and oxytocin-producing magnocellular neurons of the supraoptic and paraventricular nucleus, which
are not affected by the Alzheimer’s disease, a significant
increase in the size of the Golgi complex with age has
been found (Lucassen et al. 1994) indicating that in the
aging and failing brain neurons may still have some capacity to increase their protein processing capability.
We found no atrophy or fragmentation of Golgi apparatus in brain neurons of the rats injected i.c.v. with
STZ. Instead, the most prominent ultrastructural feature
of these cells was the apparent expansion of the trans
part of the Golgi complex. A similar phenomenon has
also been seen in cortical neurons following treatment
with 2-deoxyglucose. Thus, in respect to Golgi complex
morphology (at least at 3 weeks following injection of
the toxin), the i.c.v. STZ model does not resemble Alzheimer’s disease. It is, however, possible that the expansion of the trans part of the Golgi is a transient
phenomenon which occurs only in pre-symptomatic
AD, transforming later in the course of this disease into
the fragmentation and atrophy of this organelle. This
possibility requires further study.
The impression of a preferential expansion of the
trans part of the Golgi complex has been confirmed by
quantitative analysis, although our morphometric data
must be interpreted cautiously. The Golgi complex is
currently viewed as a series of four, or at least three
subcompartments termed cis-, medial-, trans-Golgi,
and trans-Golgi Network (TGN) (Glick 2000), or
cis-Golgi network (CGN), stack of Golgi cisternae
(STN) and TGN (del Valle et al. 1999). It remains unsettled whether these subcompartments are stable or
formed through a continuous dynamic outgrowth and
maturation of the Golgi cisternae, although recent evidence seems to favor the second model (Glick 2000). In
either case the Golgi complex consists of a series of
subcompartments which spread in an orderly fashion
between its cis-face neighboring endoplasmatic reticulum and its trans- face directed toward the outer plasma
membrane and differ from each other in biochemical
composition of the membranes and physicochemical
milieu inside the cisterns. The subcompartments may be
localized with dedicated cytochemical techniques such
as, lectinocytochemistry (Pavelka and Ellinger 1991),
but in our morphometric analysis we did not use any
such method. Instead, each Golgi area was divided into
cis and trans parts on the basis of the gross structural criteria only. One may argue that such an approach provides data of a limited value. Interestingly, however, in
experiments with cultured NRK (normal rat kidney)
cells depletion of the cellular ATP pool to 15-25% of the
control has been reported to result in a specific disassembly of the CGN (i.e., the part neighboring
endoplasmic reticulum), whereas the other parts of the
Golgi appeared energy-depletion insensitive (del Valle
et al. 1999). When the same cell system was exposed to
i.c.v. streptozotocin and Golgi morphology 487
20°C to block the transport of proteins out of the Golgi
complex, the dominant feature of Golgi structure were
the changes in the trans-most cisternae (Ladinsky et al.
2002). These data show that changes in gross Golgi
morphology can be stimulus-specific and, depending on
the stimulus, either the cis or trans part of the Golgi
complex can be preferentially affected. Our morphometric evaluation (which was performed blindly) unequivocally supports the impression that treatment with
both i.c.v. STZ and (to a lesser extent) i.v. 2-deoxyglucose produce preferential enlargement of the trans
part of the Golgi complex. A common mechanism may
be employed by which STZ as well, as 2-DG influence
Golgi morphology. This issue is discussed in the
accompanying paper (Grieb et al. 2004).
A possibility that the relative enlargement of the
trans part of the Golgi complex may influence
proteolytic processing of b-APP may also be considered. This process is compartmentalized along the secretory pathway of neurons so that distinct populations
of Abeta amyloid peptides are generated in the
endoplasmic reticulum and in the Golgi complex
(Greenfield et al. 1999). It has recently been suggested
that a very highly amyloidogenic truncated amyloid
species Abeta(11-40/42), which accumulates in Alzheimer’s brains, is preferentially generated in the
trans-Golgi complex (Huse et al. 2002). The expansion
of the distal part of the neuronal secretory pathway in response to brain insulin receptors’ desensitization may
provide for a relatively longer residence of b-APP in the
trans-Golgi environment. This may result in increased
generation of the truncated, particularly amyloidogenic
form of Abeta. Such a mechanism could provide a link
between the development of brain insulin receptor desensitization and the stimulation of amyloidogenesis
during the early phase of the Alzheimer’s disease.
CONCLUSIONS
Three weeks after double intracerebroventricular injection of streptozotocin in rats a significant expansion
of the trans-Golgi part of the secretory pathway in cortical neurons was observed. This effect was similar, but
more pronounced than the effect of intravenous
2-deoxyglucose seen 7.5 h after injection. Such a picture is very different from Golgi complex atrophy and
fragmentation seen in Alzheimer’s brains, but it may
represent an early response to insulin receptor desensitization. Considering that a very highly amyloidogenic
truncated amyloid species Abeta(11-40/42) is preferentially generated in the trans-Golgi complex, expansion
of the distal part of the intracellular secretory pathway
may facilitate amyloidogenesis.
ACKNOWLEDGEMENT
The study was supported by the State Committee for
Scientific Research (KBN) project no. 4P05A 041 17
granted to the late professor Miroslaw J. Mossakowski
to whom the authors are indebted for his creativity and
expertise in the early stage of the work.
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Received 10 August 2003, accepted 18 February 2004